Dr. B Notes (090513):
determine best PDB to use for Homology Model - see Target Discovery protocol
create a Homology model using ICM - see GoogleDocs/Protocols/VirtualScreening/HomologyModel/
Instructions ProMol Project Homology Models v6
Serine/threonine protein phosphatase PP1 (Plasmodium falciparum)
Etiologic Risk Group (see link below): Risk Group 2 (RG2) - Parasitic Agents
Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Plasmodium falciparum is a parasite that is one of the most common causes of malaria. In 2002, it was estimated that 2.2 billion people were exposed to malaria, resulting in approximately 515 million clinical attacks of this disease. The majority (~70%) of incidence of this disease in 2002 occurred in Africa, with a substantial incidence rate (~25%) in Southeast Asia [1]. A global map depicting the endemicity of malaria in 2007 showed high endemicity in Africa, low endemicity with pockets of mid to high endemicity in Central/Southeast Asia, and uniformly low endemicity in the Americas [2].
Because malaria is generally concentrated in lower income countries, the ideal treatment option for malaria would be cheap, single-dose and have minimal side effects. However, the current treatment options fall short of meeting this criteria. For decades, choroquine (CQ) was the main treatment option for malaria; however, as years passed, CQ-resistant malaria developed. Today, CQ is no longer a viable treatment option for most instances of malaria. Other drugs used in malaria treatment have serious side effects. As such, there is a pronounced need for novel anti-malarial drugs [3].
Essentiality of this protein:
Serine and threonine have similar side-chain compositions and thus can be phosphorylated by a single enzyme called serine/threonine protein kinase. The addition of the phosphate group can be reversed by an enzyme called serine/threonine phosphatase. The removal of the phosphate group helps to regulate many cellular pathways involved in cell proliferation, programmed cell death (apoptosis), embryonic development, and cell differentiation.
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Reagent availability: Reagent: MAL13P1.274; Type: cloned gene; Source: BRENDA Notes: A gene with this EC number or name or sequence has been cloned from Plasmodium falciparum ( 1 );
Reagent: MAL13P1.274; Type: purified protein; Source: BRENDA Notes: A protein with this EC number or name or sequence has been purified from Plasmodium falciparum ( 1 );
-- List cost and quantity of substrate reagents and supplier
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Homology Model for STPP PP1
---- Query Coverage:
---- Max % Identities: 85%
---- % Positives: 95%
---- Chain used for homology: 3v4y_G
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells):
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MALEIDIDNVISKLIEVRGTRPGKNVNLTENEIKILCLSSREIFLNQPILLELEAPIKIC GDIHGQFYDLLRLFEYGGFPPDANYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLR GNHECASINRIYGFYDECKRRYSVKLWKTFIDCFNCLPVAAIIDEKIFCMHGGLSPELNN MEQIRKITRPTDVPDNGLLCDLLWSDPEKEINGWGENDRGVSFTFGQDVVHNFLRKHELD LICRAHQVVEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPVEKK KAAN
*length of your protein in Amino Acids: 304 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 76889
Molar Extinction coefficient of your protein at 280 nm wavelength:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGGCGCTGGAAATCGACATCGACAACGTGATCTCCAAACTGATCGAAGTTCGTGGTACC
61 CGTCCAGGCAAGAACGTTAACCTGACCGAGAATGAGATCAAAATTCTGTGCCTGTCTTCT
121 CGTGAAATCTTTCTCAATCAGCCGATCCTGCTCGAACTGGAAGCGCCGATTAAGATCTGC
181 GGTGATATCCATGGTCAGTTCTACGACCTGCTCCGTCTGTTCGAATATGGTGGTTTCCCA
241 CCGGACGCGAATTATCTGTTCCTGGGTGACTACGTTGACCGCGGCAAACAGTCTCTGGAG
301 ACCATCTGTCTGCTCCTGGCGTACAAAATCAAATATCCGGAAAATTTCTTCCTGCTGCGT
361 GGTAACCATGAATGCGCGTCTATCAACCGTATCTACGGTTTCTATGACGAATGCAAACGT
421 CGTTACTCTGTTAAACTGTGGAAGACCTTCATCGACTGCTTCAACTGCCTCCCGGTTGCG
481 GCGATTATCGACGAAAAAATCTTCTGTATGCACGGTGGTCTCTCTCCGGAACTGAACAAC
541 ATGGAACAAATCCGTAAAATCACCCGCCCAACGGACGTTCCTGACAACGGTCTGCTGTGC
601 GACCTCCTGTGGTCTGACCCTGAAAAAGAAATCAACGGTTGGGGTGAAAACGACCGTGGT
661 GTTTCTTTCACCTTCGGTCAAGACGTCGTGCACAATTTTCTGCGTAAACATGAACTCGAC
721 CTGATCTGCCGTGCGCATCAGGTTGTTGAAGACGGTTACGAATTCTTCGCTAAGCGTCAG
781 CTCGTTACCCTGTTCTCTGCGCCGAACTACTGTGGTGAGTTCGATAACGCGGGTGCGATG
841 ATGTCTGTTGACGAAACCCTGATGTGCTCTTTCCAGATTCTGAAGCCGGTTGAAAAGAAG
901 AAGGCGGCGAACTAA
*GC% Content for gene (codon optimized): 50%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References
[1] Snow, R.W.; Guerra, C.A.; Noor, A.M.; Myint, H.Y.; Hay, S.I., The global distribution of clinical episodes of plasmodium falciparum malaria. Nature2005, 434, (7030), 214-217.
determine best PDB to use for Homology Model - see Target Discovery protocol
create a Homology model using ICM - see GoogleDocs/Protocols/VirtualScreening/HomologyModel/
Instructions ProMol Project Homology Models v6
Serine/threonine protein phosphatase PP1 (Plasmodium falciparum)
*Target (protein/gene name): serine/threonine protein phosphatase PP1http://www.tdrtargets.org/targets/view?gene_id=2475
http://plasmodb.org/plasmo/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=PF14_0142
*NCBI Gene # or RefSeq#:
PF3D7_1414400
*Protein ID (NP or XP #) or Wolbachia#:
*Organism (including strain):
Plasmodium falciparum 3D7
Etiologic Risk Group (see link below): Risk Group 2 (RG2) - Parasitic Agents
Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium falciparum is a parasite that is one of the most common causes of malaria. In 2002, it was estimated that 2.2 billion people were exposed to malaria, resulting in approximately 515 million clinical attacks of this disease. The majority (~70%) of incidence of this disease in 2002 occurred in Africa, with a substantial incidence rate (~25%) in Southeast Asia [1]. A global map depicting the endemicity of malaria in 2007 showed high endemicity in Africa, low endemicity with pockets of mid to high endemicity in Central/Southeast Asia, and uniformly low endemicity in the Americas [2].
Because malaria is generally concentrated in lower income countries, the ideal treatment option for malaria would be cheap, single-dose and have minimal side effects. However, the current treatment options fall short of meeting this criteria. For decades, choroquine (CQ) was the main treatment option for malaria; however, as years passed, CQ-resistant malaria developed. Today, CQ is no longer a viable treatment option for most instances of malaria. Other drugs used in malaria treatment have serious side effects. As such, there is a pronounced need for novel anti-malarial drugs [3].
Essentiality of this protein:
Serine and threonine have similar side-chain compositions and thus can be phosphorylated by a single enzyme called serine/threonine protein kinase. The addition of the phosphate group can be reversed by an enzyme called serine/threonine phosphatase. The removal of the phosphate group helps to regulate many cellular pathways involved in cell proliferation, programmed cell death (apoptosis), embryonic development, and cell differentiation.
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb927.10.13670 (OG4_11778); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Complex of proteins?:
no
Druggable Target:
0.6 (yes)
*EC#: 3.1.3.16
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
-- Show screenshot of BRENDA enzyme mechanism schematic
Assay: Assay for Calcineurin (3.1.3.16 ); Source: Sigma-Aldrich
Assay: 4-nitrophenyl phosphate (PNPP)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf
Reagent availability:
Reagent: MAL13P1.274; Type: cloned gene; Source: BRENDA
Notes: A gene with this EC number or name or sequence has been cloned from Plasmodium falciparum ( 1 );
Reagent: MAL13P1.274; Type: purified protein; Source: BRENDA
Notes: A protein with this EC number or name or sequence has been purified from Plasmodium falciparum ( 1 );
-- List cost and quantity of substrate reagents and supplier
Structure Available (PDB or Homology model)
http://www.ncbi.nlm.nih.gov/protein/410562615?report=genbank&log$=prottop&blast_rank=3&RID=8JUAP21S01R
http://www.rcsb.org/pdb/explore/explore.do?pdbId=3v4y
Newest Blastp homology result (Sept. 4, 2013):
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities: 85%
---- % Positives: 95%
---- Chain used for homology: 3v4y_G
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells):
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MALEIDIDNVISKLIEVRGTRPGKNVNLTENEIKILCLSSREIFLNQPILLELEAPIKIC GDIHGQFYDLLRLFEYGGFPPDANYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLR GNHECASINRIYGFYDECKRRYSVKLWKTFIDCFNCLPVAAIIDEKIFCMHGGLSPELNN MEQIRKITRPTDVPDNGLLCDLLWSDPEKEINGWGENDRGVSFTFGQDVVHNFLRKHELD LICRAHQVVEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPVEKK KAAN
*length of your protein in Amino Acids: 304 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 76889
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene: 50%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGGCGCTGGAAATCGACATCGACAACGTGATCTCCAAACTGATCGAAGTTCGTGGTACC
61 CGTCCAGGCAAGAACGTTAACCTGACCGAGAATGAGATCAAAATTCTGTGCCTGTCTTCT
121 CGTGAAATCTTTCTCAATCAGCCGATCCTGCTCGAACTGGAAGCGCCGATTAAGATCTGC
181 GGTGATATCCATGGTCAGTTCTACGACCTGCTCCGTCTGTTCGAATATGGTGGTTTCCCA
241 CCGGACGCGAATTATCTGTTCCTGGGTGACTACGTTGACCGCGGCAAACAGTCTCTGGAG
301 ACCATCTGTCTGCTCCTGGCGTACAAAATCAAATATCCGGAAAATTTCTTCCTGCTGCGT
361 GGTAACCATGAATGCGCGTCTATCAACCGTATCTACGGTTTCTATGACGAATGCAAACGT
421 CGTTACTCTGTTAAACTGTGGAAGACCTTCATCGACTGCTTCAACTGCCTCCCGGTTGCG
481 GCGATTATCGACGAAAAAATCTTCTGTATGCACGGTGGTCTCTCTCCGGAACTGAACAAC
541 ATGGAACAAATCCGTAAAATCACCCGCCCAACGGACGTTCCTGACAACGGTCTGCTGTGC
601 GACCTCCTGTGGTCTGACCCTGAAAAAGAAATCAACGGTTGGGGTGAAAACGACCGTGGT
661 GTTTCTTTCACCTTCGGTCAAGACGTCGTGCACAATTTTCTGCGTAAACATGAACTCGAC
721 CTGATCTGCCGTGCGCATCAGGTTGTTGAAGACGGTTACGAATTCTTCGCTAAGCGTCAG
781 CTCGTTACCCTGTTCTCTGCGCCGAACTACTGTGGTGAGTTCGATAACGCGGGTGCGATG
841 ATGTCTGTTGACGAAACCCTGATGTGCTCTTTCCAGATTCTGAAGCCGGTTGAAAAGAAG
901 AAGGCGGCGAACTAA
*GC% Content for gene (codon optimized): 50%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References
[1] Snow, R.W.; Guerra, C.A.; Noor, A.M.; Myint, H.Y.; Hay, S.I., The global distribution of clinical episodes of plasmodium falciparum malaria. Nature 2005, 434, (7030), 214-217.
[2] Hay, S.I.; Guerra, C.A.; Gething, P.W.; Patil, A.P.; Tatem, A.J.; Noor, A.M.; Kabaria, A.M.; Manh, B.H.; Elyazar, I.R.F.; Brooker, S.; Smith, D.L.; Moyeed, R.A.; Snow, R.W., A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med 2009, 6, (3).
[3] Bell, D.J.; Molyneux, M.E., Treatment of childhood plasmodium falciparum malaria: Current challenges. Expert Review of Anti-Infective Therapy 2007, 5, (1), 141.